Summary Osteoporosis has a devastating impact on the health and quality of life of women and men. Several FDA approved drugs are available for the treatment of osteoporosis. However, in the US a low and declining number of patients at high risk for fractures do not receive adequate treatment because of the cost and the side effects of currently available drugs. This trend has generated ?a crisis? in the treatment osteoporosis. Thus, there is a critical need to identify inexpensive, safe and effective interventions for both the prevention and treatment of osteoporosis. Interestingly, there is strong evidence that the gut microbiome regulates bone homeostasis in health and disease, and that probiotics protect against bone loss. In 2016, we published in JCI that sex steroid depleted germ-free mice do not undergo trabecular bone loss, demonstrating a role of the microbiota in the bone loss induced by sex steroid deprivation. We showed that Lactobacillus rhamnosus GG (LGG) and VSL#3 probiotics completely protected ovariectomized (ovx) mice from bone loss by decreasing bone resorption. Furthermore, these probiotics increased bone mass in estrogen replete controls by stimulating bone formation. New preliminary data confirm that LGG and VSL#3 exert bone anti-catabolic effects in ovx mice, as well as bone anabolic effects in estrogen replete controls. Based on preliminary data, our central hypothesis is that probiotics stimulate bone formation by generating the short-chain fatty acid (SCAFA) butyrate in the gut. Butyrate has been shown to induce regulatory T cell (Treg) expansion in the intestine. We show preliminary data that probiotics, or direct feeding of butyrate, also induces the expansion of the Treg population in the bone marrow (BM). Mechanistically, we show that Treg expansion induces the release of the osteogenic Wnt ligand Wnt10b by BM CD8+ T cells. Wnt10b then activates Wnt signaling in osteoblastic cells, leading to increased bone formation. Our published studies show that probiotics prevent bone loss in ovx mice by reversing an increase in gut permeability induced by ovx. We now hypothesize that probiotics block bone resorption in ovx mice by decreasing gut permeability via activation of ERK dependent signaling pathways in gut epithelial cells, leading to lower intestinal inflammation. We also hypothesize that probiotics block the trafficking of activated immune cells from the gut to the BM. Finally, we show that probiotics can change gut microbiome diversity, suggesting that probiotics prevent inflammation and bone loss in ovx mice by restoring gut microbiota diversity. Our hypotheses will be tested in 3 specific aims: 1) To determine if LGG and VSL#3 probiotics stimulate bone formation in intact mice via a SCFAs/Treg/ CD8+T cells/Wnt10b dependent mechanism, 2) To identify the molecular events at the gut epithelium that mediate probiotic protection against bone loss following sex steroid depletion, and 3) To determine whether probiotics mechanistically influence bone metabolism by preserving a eubiotic microbiome diversity. The outcomes of these studies are relevant to the mission of NIH by addressing novel and inexpensive interventions for osteoporosis.